Biodiesel and poly‐unsaturated fatty acids production from algae and crop plants – a rapid and comprehensive workflow for lipid analysis

Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC‐MS) is a widely used technique in biodiesel/bioproduct (e.g. poly‐unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC‐EI‐MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC‐MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC‐FI‐MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high‐quality bioproducts for nutrition such as PUFAs.     

Usutu virus activity in Austria, 2001-2002

Usutu virus (USUV), a member of the mosquito-borne clade within the Flaviviridae family, was responsible for avian mortality in Austria in 2001. In 2002, the virus continued to kill birds, predominantly blackbirds. High numbers of avian deaths were recorded within the city of Vienna and in surrounding districts of the federal state of Lower Austria, while single die-offs were noticed in the federal states of Styria and Burgenland. A total of 72 birds were submitted for laboratory examination, 30 of which tested positive for USUV by immunohistochemistry and/or polymerase chain reaction. Laboratory-confirmed cases of USUV infection originated from the federal states of Vienna and Lower Austria only. The data show that (i) USUV has managed to overwinter and has been able to establish a transmission cycle in Austria, (ii) the virus seems to have become a resident pathogen of Austria with a tendency to spread to other geographic areas, and (iii) the surveillance of dead blackbirds is a useful sentinel system for monitoring USUV activity.     

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti‐depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity‐dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N‐phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N‐phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.     

Bronchopulmonary aspergillosis in lamb

In a 7 month old female lamb from a pulmonary and intestinal worm infested flock in Southern Bohemia, morbid anatomical and histological changes were seen which suggested aspergillosis. Aspergillus fumigatus Fresenius was found and proved by both microscopic examination and culture.

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Zusammenfassung In einem 7 Monate alten, weiblichen Schaf von einem Herd von Südböhmen, das Lungenleiden und intestinale Wurminfektion zeigte, sind pathologisch-anatomisch und histologisch Veränderungen gefunden worden, die auf Aspergillosis hinwiesen.Aspergillus fumigatus Fres. ist mikroskopisch und kulturell nachgewiesen worden.

     

Plastic and genetic responses of a common sedge to warming have contrasting effects on carbon cycle processes

Climate warming affects plant physiology through genetic adaptation and phenotypic plasticity, but little is known about how these mechanisms influence ecosystem processes. We used three elevation gradients and a reciprocal transplant experiment to show that temperature causes genetic change in the sedge Eriophorum vaginatum. We demonstrate that plants originating from warmer climate produce fewer secondary compounds, grow faster and accelerate carbon dioxide (CO₂) release to the atmosphere. However, warmer climate also caused plasticity in E. vaginatum, inhibiting nitrogen metabolism, photosynthesis and growth and slowing CO₂ release into the atmosphere. Genetic differentiation and plasticity in E. vaginatum thus had opposing effects on CO₂ fluxes, suggesting that warming over many generations may buffer, or reverse, the short‐term influence of this species over carbon cycle processes. Our findings demonstrate the capacity for plant evolution to impact ecosystem processes, and reveal a further mechanism through which plants will shape ecosystem responses to climate change.     

Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Preparation and evaluation of porcine sera against human immunoglobulins

Thirty-eight pigs were immunized with normal human γG-, γA- and γM-immunoglobulins, fragments of γG-immunoglobulin (Fab, Fc+F'c), type K and L Bence-Jones proteins and other antigens containing immunoglobulins or their fragments. The pigs formed antibodies readily against the heavy γ, α, μ and δ chains and also against η chains. No antibodies whatsoever were formed against the surface determinants of λ chains; the antibodies present reacted only with hidden determinants of these chains. In isolated cases, antibodies against the Fd fragments of γG-immunoglobulin were also identified. A quantitative precipitation test showed that porcine antibodies are of the “R”, precipitin, type.